Hearing loss affects millions of people worldwide and often results from death of the sensory hair cells in the inner ear. Hair cells are lost due to noise exposure, drug damage, and progressively during the aging process. The overall goal of the proposed research is to further understand the intracellular signaling pathways responsible for hair cell death due to drug damage, specifically damage caused by the aminoglycoside antibiotics. This research will use the zebrafish lateral line, a powerful in vivo model system for real-time hair cell death and protection studies. Our previous research has demonstrated that the closely related aminoglycosides neomycin and gentamicin damage hair cells via at least two distinct and partially overlapping cell death processes. The specific goal of the proposed research is to understand the intracellular events that differentiate these cell death processes with a focus on the multifaceted role of the tumor suppressor protein p53 during hair cell death in this system. This research will test the hypothesis that aminoglycoside-induced hair cell death in the zebrafish lateral line is dependent on diverse p53 functions, with the acute damage caused by neomycin or short-term gentamicin exposure requiring direct activity of p53 at the mitochondria, and the slower phase of gentamicin-induced damage dependent on p53 transcriptional activity in the nucleus. Specifically, the proposed project will 1) test the requirement for p53 in aminoglycoside-induced hair cell death, 2) characterize the involvement of direct p53 mitochondrial activity during aminoglycoside exposure, and 3) test the requirement for p53 nuclear activity during aminoglycoside-induced hair cell damage. A more thorough understanding of the specific cell death pathways activated by different hair cell toxins will allow us to more directly target potential protective therapies, allowing for hearing preservation in patients treated with ototoxic drugs.